The present invention relates to the production and screening of expression libraries for enzyme activity and, more particularly, to obtaining selected DNA from DNA of a microorganism and to screening of an expression library for enzyme activity which is produced from selected DNA.
Industry has recognized the need for new enzymes for a wide variety of industrial applications. As a result, a variety of microorganisms have been screened to ascertain whether such microorganisms have a desired enzyme activity. If such microorganisms do have the desired enzyme activity, the enzyme is then recovered from them.
The present invention provides a novel approach for obtaining enzymes for further use, for example, for a wide variety of industrial applications, for medical applications, for packaging into kits for use as research reagents, etc. In accordance with the present invention, recombinant enzymes are generated from microorganisms and are classified by various enzyme characteristics.
More particularly, one aspect of the present invention provides a process for identifying clones having a specified enzyme activity, which process comprises:
screening for said specified enzyme activity in a library of clones prepared by
(i) selectively isolating target DNA, from DNA derived from at least one microorganism, by use of at least one probe DNA comprising at least a portion of a DNA sequence encoding an enzyme having the specified enzyme activity; and
(ii) transforming a host with isolated target DNA to produce a library of clones which are screened, preferably for the specified enzyme activity, using an activity library screening or nucleic acid library screening protocol.
In a preferred embodiment of this aspect, DNA obtained from at least one microorganism is selected by recovering from the DNA, DNA which spcifically binds, such as by hybridization, to a probe DNA sequence. The DNA obtained from the microorganism or microorganisms can be genomic DNA or genomic gene library DNA. One could even use DNA prepared for vector ligation, for instance. The probe may be directly or indirectly bound to a solid phase by which it is separated from the DNA which is not hybridized or otherwise specifically bound to the probe. The process can also include releasing DNA from said probe after recovering said hybridized or otherwise bound DNA and amplifying the DNA so released.
The invention also provides for screening of the expression libraries for gene cluster protein product(s) and, more particularly, to obtaining selected gene clusters from DNA of a prokaryote or eukaryote and to screening of an expression library for a desired activity of a protein of related activity(ies) of a family of proteins which results from expression of the selected gene cluster DNA of interest.
More particularly, one embodiment of this aspect provides a process for identifying clones having a specified protein(s) activity, which process comprises screening for said specified enzyme activity in the library of clones prepared by (i) selectively isolating target gene cluster DNA, from DNA derived from at least one organism, by use of at least one probe polynucleotide comprising at least a portion of a polynucleotide sequence complementary to a DNA sequence encoding the protein(s) having the specified activity of interest; and (ii) transforming a host with isolated target gene cluster DNA to produce a library of such clones which are screened for the specified activity of interest. For example, if one is using DNA in a lambda vector one could package the DNA and infect cells via this route.
In a particular embodiment of this aspect, gene cluster DNA obtained from the genomic DNA of the organism(s) is selected by recovering from the DNA, DNA which specifically binds, such as by hybridization, to a probe DNA sequence. The polynucleotide probe may be directly or indirectly bound to a solid phase by which it is separated from the DNA which is not hybridized or otherwise specifically bound to the probe. This embodiment of this aspect of the process of the invention can also include releasing bound DNA from said probe after recovering said hybridized or otherwise bound DNA and amplifying the DNA so released.
These and other aspects of the present invention will be apparent to those skilled in the art from the teachings herein.